The present invention relates to a rapid, sensitive, simple, and cost-effective spectrophotometric method of detecting and quantifying mycolic acid in a mycolic acid-fuschin dye complex with absorbance maxima ranging between 490-500 nm in the presence of various test compounds, for screening mycolic acid biosynthesis inhibitors useful as anti-microbial agents and a diagnostic kit thereof comprising basic fuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml, phenol and 95% ethanol in the ratio ranging between 1:4 to 2:1 (v/v), and phenol and distilled water in the ratio ranging between 1:14 to 1:25.
Tuberculosis caused by Mycobacterium tuberculosis is a public health problem, which has increased in importance during the last two decades, due in part to the increasing number of cases caused by the association of acquired immunodeficiency syndrome (AIDS) and the appearance of multiple drug-resistant strains. Other mycobacteria which are often indistinguishable from tuberculosis have also increased.
The current method of detection is by determining the mycolic acid patterns from clinical isolates of sputum, cerebrospinal fluid, bronchial washing, corneal ulcer, and bone marrow, as well as from acid-fast stain smear-positive clinical specimens. Standardized mycolic acid extraction methods are used to ensure the maximal extraction of mycolic acid derivatives to enhance the sensitivity of the method. Different chromatographic columns are used to identify the species of mycolic acid. The immediate detection of bacteria containing mycolic acid is through acid fast staining and detection through the microscope. Lipid-rich cell walls are not permeable by ordinary stains.
The stain consisting of a basic dye (fuchsin) and phenol (a lipid solvent) is used for the purpose. Phenol partially solubilizes the cell wall and allows fuchsin to penetrate the wall and bind to mycolic acid.
After fuchsin is incorporated, it is resistant to decolorization even after exposure to acid alcohol, a property that characterizes mycobacteria and that can be performed on unprocessed clinical specimens, concentrated specimens, or cultures.
This principle is widely used to identify bacteria producing mycolic acid with the help of a microscope. But an improved system based on visual detection of the mycolic acid is the need of the hour to screen out potential mycolic acid biosynthesis inhibiting compounds from a vast array of plant compounds.
The detection and quantification procedure can be made more accurate by using the help of spectrophotometer. The amount of inhibition can be quantified simply through optical density measurement at a particular wavelength. This does not necessitate the complex, high cost technologies like IR, NMR, GC spectroscopy for the detection, which are currently being used.
The amount of mycolic acid which cannot be quantified through acid fast staining followed by microscopic detection can be quantified by systematic extraction and dye binding followed by spectrophotometric analysis. Keeping this in mind, a simple, rapid, cost effective procedure kit was devised to detect and quantify the mycolic acid which in turn resulted in a high efficiency screen to identify potential inhibitors of mycolic acid biosynthesis.
The protocols for extraction, isolation of mycolic acid and detection of bacteria-producing mycolic acid through acid fast staining are known in the art. But the system as a whole is inefficient and requires a microscope to detect the presence of the bacteria-producing mycolic acid.
Similarly, extraction, isolation and characterization of mycolic acid is carried out through different methods of chromatography and IR, NMR spectroscopy. The emphasis of the present invention was to generate a system to screen a large amount of plant extracts and compounds with potential mycolic acid biosynthesis inhibiting activity, rapidly and with a low cost.
The main object of the present invention is to develop a quick, simple, sensitive, cost-effective method of identifying mycolic acid.
Another main object of the present invention is to develop a quick, simple, sensitive, cost-effective method of quantifying mycolic acid.
A further object of the present invention is to develop a quick, simple, sensitive, cost-effective spectrophotometric method of identifying and quantifying mycolic acid.
Yet another object of the present invention is to use carbol-fuschin dye and mycolic acid complex to develop a quick, simple, sensitive, cost-effective spectrophotometric method of identifying and quantifying mycolic acid.
Still another object of the present invention is to develop a method of screening test compounds for their mycolic acid inhibitory properties.
Still another object of the present invention is to develop a diagnostic kit for the detection of mycolic acid by spectrophotometry.
The present invention relates to a rapid, sensitive, simple, and cost-effective spectrophotometric method of detecting and quantifying mycolic acid in a mycolic acid-fuschin dye complex with absorbance maxima ranging between 490-500 nm in the presence of various test compounds, for screening mycolic acid biosynthesis inhibitors useful as anti-microbial agents and a diagnostic kit thereof comprising basic fuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml, phenol and 95% ethanol in the ratio ranging between 1:4 to 2:1 (v/v), and phenol and distilled water in the ratio ranging between 1:14 to 1:25.
Accordingly, the present invention relates to a rapid, sensitive, simple, and cost-effective spectrophotometric method of detecting and quantifying mycolic acid in a mycolic acid-fuschin dye complex with absorbance maxima ranging between 490-500 nm in the presence of various test compounds, for screening mycolic acid biosynthesis inhibitors useful as anti-microbial agents and a diagnostic kit thereof comprising basic fuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml, phenol and 95% ethanol in the ratio ranging between 1:4 to 2:1 (v/v), and phenol and distilled water in the ratio ranging between 1:14 to 1:25.
According to an embodiment of the present invention there is provided a rapid, simple, sensitive, and cost effective method of screening mycolic acid biosynthesis inhibitors useful as anti-microbial agents by quantifying the amount of mycolic acid produced by bacteria in the presence and absence of test compound or extract.
According to another embodiment of the present invention, there is provided the step growing separate cultures of the specified bacteria in the presence and absence of the specified compound or extract for time duration ranging between 40-60 hours.
According to yet another embodiment of the present invention, a lyophilizing bacterial pellet is produced.
According to still another embodiment of the present invention, there is provided the step of extracting mycolic acid by a conventional method.
According to still another embodiment of the present invention, there is provided the step of dissolving extracted mycolic acid extract in hexane to obtain mycolic acid solution.
According to still another embodiment of the present invention, there is provided the step of adding the specified solution to carbol-fuschin dye in the ratio ranging between 1:4 to 4:1 (v/v).
According to still another embodiment of the present invention, there is provided the step of shaking the product of the above step vigorously to obtain a pink color mycolic acid-dye complex as an upper layer.
According to still another embodiment of the present invention, there is provided the step of quantifying the specified mycolic acid spectrophotometrically at a wavelength ranging between 490-500 nm.
According to still another embodiment of the present invention, there is provided the step of determining the degree of inhibition of mycolic acid biosynthesis in the compound-treated bacterial culture.
In still another embodiment of the present invention, wherein quantifying mycolic acid level spectrophotometrically at wavelength preferably ranging between 494-496 nanometer.
According to still another embodiment of the present invention, the ratio of carbol fuchsin dye to the specified extract is preferably 1:1 (v/v).
According to still another embodiment of the present invention, the carbol fuschin consists of basic fuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml, phenol, 95% ethanol, and distilled water, the constituents being provided in the ratio, phenol:ethanol in ratio ranging between 1:4 to 2:1 (v/v), and phenol: distilled water in the ratio ranging between 1:25 to 1:14 (v/v).
According to still another embodiment of the present invention, the specified test compound or extract with inhibitory activity is added at concentrations ranging between {fraction (1/25)} to xc2xd of minimum inhibitory concentration (MIC).
According to still another embodiment of the present invention, the specified anti-microbial agents are developed against microbes comprising Mycobacterium, Corynebacterium, and Nocardia. 
According to still another embodiment of the present invention, the specified method is used to screen for inhibitors selected from a group comprising synthetic, semi-synthetic, natural compounds, and extracts.
According to still another embodiment of the present invention, the intensity of color increases with an increase in the concentration of mycolic acid.
According to still another embodiment of the present invention, the specified method works for mycolic acid from all sources.
According to a further embodiment of the present invention, a diagnostic kit is provided that is useful for identifying potential anti-microbial drugs against mycolic acid producing microbes, the kit comprising carbol fuschin, methanol, toluene, hexane, concentrated sulphuric acid, a bacterial pellet, and a test compound or extract, with the constituents in the ratio: methanol:toluene ranging between 1:3 to 3:1 (v/v), methanol:hexane ranging between 8:1 to 2:1 (v/v), concentrated sulphuric acid:hexane ranging between 1:8 to 1:2 (v/v), and carbol fuschin:hexane extract ranging between 1:4 to 4:1 (v/v), with methanol, toluene, and concentrated sulphuric acid of above-mentioned ratio added into lyophilized bacterial pellet with final concentration of the same ranging between 0.1-3.0 gm/100 ml.
According to still another embodiment of the present invention, the carbol fuschin consists of basic fuschin dye in the concentration ranging between 0.1-1.0 gm/100 ml, phenol, 95% ethanol, and distilled water, the constituents being provided in the ratio: phenol:ethanol in ratio ranging between 1:4 to 2:1 (v/v), and phenol:distilled water in the ratio ranging between 1:14 to 1:25 (v/v).
According to still another embodiment of the present invention, the specified anti-microbial agents are developed against microbes comprising Mycobacterium, Corynebacterium, and Nocardia. 
According to still another embodiment of the present invention, the specified test compound or extract with inhibitory activity is added at concentrations ranging between {fraction (1/25)} to {fraction (1/2)} of minimum inhibitory (MIC).
According to another embodiment of the present invention, in acid fast staining, a characteristic feature of the genus Mycobacteria causes mycolic acid to bind with carbol fuchsin dye to give the pink colour of the acid fast bacilli.
According to yet another embodiment of the present invention, on the basis of this principle a spectrophotometric assay procedure was developed, in which the hexane extract of mycolic acid, when kept in a screw cap vial along with carbol fuchsin dye, hexane fraction forms upper colorless layer and the dye forms the lower pink layer, but as the vial is mixed vigorously the carbol fuschin dye binds to mycolic acid present in the hexane layer and forms an upper pink layer the intensity of which increases and a lower transparent layer in which intensity of pink colour decreases depending on the amount of mycolic acid present in the upper hexane layer.
According to still another embodiment of the present invention, during vigorous mixing of dye with the hexane layer, there is a binding to mycolic acid to form a complex and the amount of dye goes into hexane layer depending upon the concentration of mycolic acid in the hexane layer, whereas normal hexane without mycolic acid does not form the complex to give the characteristic upper pink layer.
According to still another embodiment of the present invention, a method is disclosed for the screening of mycolic acid biosynthesis inhibitors. More particularly the method is used to detect and quantify the relative amount of mycolic acid in a mixture or a solution or a culture leading to the identification of inhibitors of mycolic acid biosynthesis, thus being useful as a screening procedure to detect potential drug compounds for inhibition of mycolic acid biosynthesis. This invention also relates to a detection kit for mycolic acid that can be used as a diagnostic kit. The present invention has direct implication in simplifying the procedure of rapid detection of compounds inhibiting the biosynthesis of mycolic acid and ultimately leading to the production of an antimicrobial drug.
According to still another embodiment of the present invention, the fact that mycolic acids in mycobacteria bind to carbol fuschin is well known. In fact, using this principal, the clinical specimens are routinely tested for the presence of mycobacterium in hospitals and diagnostic labs but with the help of a microscope, which is a laborious procedure. Moreover, the microscope method cannot be used for other applications such as drug screening where quantification of mycolic acid production by mycobacterium is a necessity.
According to still another embodiment of the present invention, the method described herein relies on the same principles but enables the quantification of the mycolic acid production and/or its inhibition in mycobacterium. The instant invention is not provided according to this principle but for the simple rapid, quantitative method for the detection of mycolic acid using a diagnostic kit and technique of spectrophotometry.
According to still another embodiment of the present invention, the present invention facilitates an improved quantification of mycolic acid presence and thus the presence of mycobacterium in any given sample. Also it facilitates an improved quantification of inhibition of mycolic acid synthesis useful in drug screening programs for the identification of mycolic acid inhibitors.